Validation of molecular diagnostic assays and quality assurance and control in the veterinary laboratory.

This chapter describes the process of validating in-house molecular assays although the principles described are equally relevant to all diagnostic assays. The best practice principles described below are based on the In Vitro Diagnostic Medical Devices Directive (IVDD) and associated documentation. Although compliance with these regulations is not required for diagnostic reagents used on animals, the principles are equally relevant to validation of all diagnostic assays, whatever their purpose.

Guidance on the development and validation of diagnostic tests that depend on nucleic acid amplification and detection.

In order to comply with national and international clinical laboratory accreditation standards (e.g. the ISO 15189, Clinical Pathology Accreditation standards) and with the joint code of practice for research, there must be a method of assessing that test methods are "fit for purpose". This document gives guidance on development and describes how a validation file is produced. A test method may be a commercial kit, an in-house assay or reagent or a set of reagents bought separately and used to prepare an in house assay. A validation file is needed for both current and new test procedures. The file may refer to data recorded in workbooks, papers and reports. Modifications to assays (including commercially available assays) necessitate either an update to the validation file or creation of a new file. This paper is intended to provide a generic framework for in-house assay development and validation of new nucleic acid amplification assays including real-time polymerase chain reaction (PCR).

Altered frequency dynamics of cervical and ocular vestibular evoked myogenic potentials in patients with Ménière's disease.

OBJECTIVE: To measure the frequency dynamics of cervical and ocular vestibular evoked myogenic potentials in healthy subjects and patients with Ménière's disease. STUDY DESIGN: A prospective cohort study. SETTING: A university teaching hospital. SUBJECTS: Eight healthy volunteers (16 ears) and 12 adult patients with unilateral Ménière's disease (8 with definite disease and 4 with probable disease) by American Academy of Otolaryngology - Head and Neck Surgery diagnostic criteria. INTERVENTIONS: Cervical and ocular vestibular evoked myogenic potentials generated by tone bursts at 250, 500, 750, 1,000, 1,500, 2,000, 3,000, and 4,000 Hz were measured in both groups. MAIN OUTCOME MEASURES: The frequency sensitivity of both the cervical and ocular vestibular evoked myogenic potentials, as evaluated by p13-n23 and n10 amplitudes in healthy ears and in ears affected and not affected by Ménière's disease. RESULTS: Cervical and ocular vestibular evoked myogenic potentials were present in all ears tested. In the healthy volunteers, the acoustic stimulus frequency at which the response amplitudes were largest was 500 Hz. This shifted to higher frequencies in patients with definite Ménière's disease for both measurements, with the effect being more pronounced for ocular vestibular evoked myogenic potentials. The shift was less marked in the probable Ménière's group and was absent in the unaffected ears of the Ménière's patients. CONCLUSION: Ménière's ears display alterations in cervical and ocular vestibular evoked myogenic potentials tuning responses with changes in the latter being more prominent. These findings indicate that the disease process affects both the otolith organs but may have an enhanced effect on the utricle. We propose that this more dominant affect may relate to the anatomical configuration of the utricle.

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates.

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.

A single-centre retrospective review of unilateral and bilateral Dysport injections in adductor spasmodic dysphonia.

We reviewed the results and side-effect profile of the Dysport preparation of botulinum toxin A (BTA) in the management of the adductor spasmodic dysphonia. We performed 272 injection episodes in 68 patients, 42 (62%) female, 26 (38%) male. A total of 116 of these injections were unilateral, and 156 were bilateral; 94% of the injections were considered to have been successful with a voice score of 2 or higher. The mean duration of effect (injection intervals) was 128.8 days in the unilateral cohort and 118.7 days in the bilateral (P > 0.05). We injected a relatively lower dose of BTA for unilateral injection episodes in our institution compared to those reported by others to produce comparable results and side-effect profiles.

A role for pericytes as microenvironmental regulators of human skin tissue regeneration.

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

Current molecular techniques for the detection of microbial pathogens.

Traditionally the detection of microbial pathogens in clinical, environmental or food samples has commonly needed the prelevation of cells by culture before the application ofthe detection strategy. This is done to increase cell number thereby overcoming problems associated with the sensitivity of classical detection strategies. However, culture-based methods have the disadvantages of taking longer, usually are more complex and require skilled personnel as well as not being able to detect viable but non cultivable microbial species. A number of molecular methods have been developed in the last 10 to 15 years to overcome these issues and to facilitate the rapid, accurate, sensitive and cost effective identification and enumeration of microorganisms which are designed to replace and/or support classical approaches to microbial detection. Amongst these new methods, ones based on the polymerase chain reaction and nucleic acid hybridization have been shown to be particularly suitable for this purpose. This review generally summarizes some of the current and emerging nucleic acid based molecular approaches for the detection, discrimination andquantification ofmicrobes in environmental, food and clinical samples and includes reference to the recently developing areas of microfluidics and nanotechnology "Lab-on-a-chip".